PROJECT SUMMARY/ABSTRACT This research program focuses on a fundamental barrier in the discovery of new drug molecules from the environment, known as the supply problem. There is no shortage of new bioactive molecules in the environment that we can use as drugs, because such molecules have been evolving for billions of years within trillions of microniches. In this program, we will (1) use both sequence analysis and directed evolution to devise ways of making promising molecules from uncultured bacteria in laboratory-grown strains, and (2) improve the discovery rate of new molecules from isolated culturable bacterial strains by determining the shared ways that their small molecule pathways are regulated, and exploiting that knowledge to turn on these pathways. 1) It is estimated that 1 trillion species of bacteria exist. The number of species that have been grown in the lab is minuscule by comparison, but that minuscule portion has given us most of the classes of antibiotics currently known as well as many other drugs. We know that we are missing an incredible amount of chemical diversity in the uncultured biosphere because we can observe the biosynthetic pathways for small molecules through culture-independent sequencing. Often these are found in the genomes of bacterial symbionts that live within another animal, such a marine invertebrate or insect. Currently, this sequence data is simply an academic curiosity because it is incredibly challenging to move pathways from uncultured symbionts to laboratory strains which might be separated by more than a billion years of evolution. We will continue to uncover important small molecule pathways in symbionts, but we will also work towards the supply of these compounds through two strategies. In the first we will devise new techniques to search for related pathways in the genomes of free- living bacteria, that might have been previously missed due to incomplete genome assembly. In the second strategy, we will use evolution to optimize protein sequences for the new host, mimicking how pathways have been horizontally transferred between different bacteria for billions of years. 2) When bacterial strains are isolated for drug discovery, most of the small molecule pathways they possess are not expressed under standard culture conditions, and we have to rely on the small subset that are produced in the lab. This is because most pathways are tightly controlled so that they are expressed under specific environmental conditions. Many small molecules made by bacteria are thought to inhibit the growth of rival species, and therefore conditional expression maximizes their impact while reducing the chance that resistance will develop. While small molecule pathways are passed between species through horizontal transfer, they become integrated into the pre-existing regulatory network of a new host. We propose to identify the global regulatory mechanisms for small molecules in a specific group of bacteria, using techniques that are generalizable, and manipulate them to produce small molecules previously inaccessible in the lab. This will overcome a major roadblock in drug discovery, allowing the full exploitation of biosynthesis in isolated strains.